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Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

Poliak S, Morales D, Croteau LP, Krawchuk D, Palmesino E, Morton S, Cloutier JF, Charron F, Dalva MB, Ackerman SL, Kao TJ, Kania A - Elife (2015)

Bottom Line: We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors.Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals.Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Columbia University, New York, United States.

ABSTRACT
During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

No MeSH data available.


Related in: MedlinePlus

Functional blocking of Neogenin has no effect on ephrin-mediated lateral LMC repulsion, quantification of Unc5c-expressing medial LMC neurons in mice and chick.(A–C) Characterization of Netrin-1/Fc stripes. Detection of Netrin-1 and Fc-Cy3 in alternating stripes by immunostaining. (D–E) Detection of lateral (EphA4+) LMC neurites of explants on C100 eA5-Fc/Fc stripes without (D) or with (E) anti-neogenin antibody treatment. Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes. Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites. Quantification of lateral (EphA4+) LMC neurites on first (pink) and second (pale) stripes expressed as a percentage of total EphA4 signals. (F) In situ hybridization for Unc5c in conjunction with immunostaining for Isl1 in e11.5 mice lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 12 sections, n=581 neurons. (G) Double in situ hybridization for Unc5c and Isl1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 6 sections, n=229 neurons. (H) Double in situ hybridization for Unc5c and Ephb1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of Ephb1+ LMC neurons which also are Unc5c+. 3 sections, n=119 neurons. LMC, lateral motor column; N, Netrin-1; eA5, ephrin-A5-Fc; error bars = SD; n.s. = not significant; statistical significance computed using Mann-Whitney U test. Scale bars: (A–E) 100 μm, (F–H) 20 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.009
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fig3s1: Functional blocking of Neogenin has no effect on ephrin-mediated lateral LMC repulsion, quantification of Unc5c-expressing medial LMC neurons in mice and chick.(A–C) Characterization of Netrin-1/Fc stripes. Detection of Netrin-1 and Fc-Cy3 in alternating stripes by immunostaining. (D–E) Detection of lateral (EphA4+) LMC neurites of explants on C100 eA5-Fc/Fc stripes without (D) or with (E) anti-neogenin antibody treatment. Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes. Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites. Quantification of lateral (EphA4+) LMC neurites on first (pink) and second (pale) stripes expressed as a percentage of total EphA4 signals. (F) In situ hybridization for Unc5c in conjunction with immunostaining for Isl1 in e11.5 mice lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 12 sections, n=581 neurons. (G) Double in situ hybridization for Unc5c and Isl1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 6 sections, n=229 neurons. (H) Double in situ hybridization for Unc5c and Ephb1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of Ephb1+ LMC neurons which also are Unc5c+. 3 sections, n=119 neurons. LMC, lateral motor column; N, Netrin-1; eA5, ephrin-A5-Fc; error bars = SD; n.s. = not significant; statistical significance computed using Mann-Whitney U test. Scale bars: (A–E) 100 μm, (F–H) 20 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.009

Mentions: In chick, the attractive Netrin receptor function has been proposed to be carried out by Neogenin, since there is no Dcc gene in this species (Phan et al., 2011). To address this possibility, we performed an Fc/N stripe preference assay incubating LMC axons in the presence of a function-blocking antibody raised against the extracellular domain of Neo1. In such experiments, LMC axon preference for Ntn1 stripes was abolished (Figure 3C, 53% on N, 47% on Fc stripes p<0.001 vs. untreated N/Fc; [Palmesino et al., 2012]), but could be rescued by electroporation of rat Dcc expression plasmid (Figure 3E; 66% on N, 34% on Fc stripes) but not with a plasmid encoding a truncated DCC (DccΔICD; Figure 3D; 48% on N, 52% on Fc stripes; p<0.001 vs. Dcc overexpression). Lateral LMC axons incubated with anti-Neo1 antibodies did not lose their sensitivity to ephrin-A5 (Figure 3—figure supplement 1). These experiments show that Neo1 mediates lateral LMC neurite growth preference on Netrin-1, and that DCC is functionally equivalent.


Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

Poliak S, Morales D, Croteau LP, Krawchuk D, Palmesino E, Morton S, Cloutier JF, Charron F, Dalva MB, Ackerman SL, Kao TJ, Kania A - Elife (2015)

Functional blocking of Neogenin has no effect on ephrin-mediated lateral LMC repulsion, quantification of Unc5c-expressing medial LMC neurons in mice and chick.(A–C) Characterization of Netrin-1/Fc stripes. Detection of Netrin-1 and Fc-Cy3 in alternating stripes by immunostaining. (D–E) Detection of lateral (EphA4+) LMC neurites of explants on C100 eA5-Fc/Fc stripes without (D) or with (E) anti-neogenin antibody treatment. Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes. Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites. Quantification of lateral (EphA4+) LMC neurites on first (pink) and second (pale) stripes expressed as a percentage of total EphA4 signals. (F) In situ hybridization for Unc5c in conjunction with immunostaining for Isl1 in e11.5 mice lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 12 sections, n=581 neurons. (G) Double in situ hybridization for Unc5c and Isl1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 6 sections, n=229 neurons. (H) Double in situ hybridization for Unc5c and Ephb1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of Ephb1+ LMC neurons which also are Unc5c+. 3 sections, n=119 neurons. LMC, lateral motor column; N, Netrin-1; eA5, ephrin-A5-Fc; error bars = SD; n.s. = not significant; statistical significance computed using Mann-Whitney U test. Scale bars: (A–E) 100 μm, (F–H) 20 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.009
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fig3s1: Functional blocking of Neogenin has no effect on ephrin-mediated lateral LMC repulsion, quantification of Unc5c-expressing medial LMC neurons in mice and chick.(A–C) Characterization of Netrin-1/Fc stripes. Detection of Netrin-1 and Fc-Cy3 in alternating stripes by immunostaining. (D–E) Detection of lateral (EphA4+) LMC neurites of explants on C100 eA5-Fc/Fc stripes without (D) or with (E) anti-neogenin antibody treatment. Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes. Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites. Quantification of lateral (EphA4+) LMC neurites on first (pink) and second (pale) stripes expressed as a percentage of total EphA4 signals. (F) In situ hybridization for Unc5c in conjunction with immunostaining for Isl1 in e11.5 mice lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 12 sections, n=581 neurons. (G) Double in situ hybridization for Unc5c and Isl1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of LMC neurons (Isl1+) which are also Unc5c+. 6 sections, n=229 neurons. (H) Double in situ hybridization for Unc5c and Ephb1 in HH stage 27 chick lumbar spinal cord. Bottom: quantification of the number of Ephb1+ LMC neurons which also are Unc5c+. 3 sections, n=119 neurons. LMC, lateral motor column; N, Netrin-1; eA5, ephrin-A5-Fc; error bars = SD; n.s. = not significant; statistical significance computed using Mann-Whitney U test. Scale bars: (A–E) 100 μm, (F–H) 20 μm.DOI:http://dx.doi.org/10.7554/eLife.10841.009
Mentions: In chick, the attractive Netrin receptor function has been proposed to be carried out by Neogenin, since there is no Dcc gene in this species (Phan et al., 2011). To address this possibility, we performed an Fc/N stripe preference assay incubating LMC axons in the presence of a function-blocking antibody raised against the extracellular domain of Neo1. In such experiments, LMC axon preference for Ntn1 stripes was abolished (Figure 3C, 53% on N, 47% on Fc stripes p<0.001 vs. untreated N/Fc; [Palmesino et al., 2012]), but could be rescued by electroporation of rat Dcc expression plasmid (Figure 3E; 66% on N, 34% on Fc stripes) but not with a plasmid encoding a truncated DCC (DccΔICD; Figure 3D; 48% on N, 52% on Fc stripes; p<0.001 vs. Dcc overexpression). Lateral LMC axons incubated with anti-Neo1 antibodies did not lose their sensitivity to ephrin-A5 (Figure 3—figure supplement 1). These experiments show that Neo1 mediates lateral LMC neurite growth preference on Netrin-1, and that DCC is functionally equivalent.

Bottom Line: We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors.Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals.Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Columbia University, New York, United States.

ABSTRACT
During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

No MeSH data available.


Related in: MedlinePlus